The long term objectives of this proposal are to understand the roles of the hepatitis B virus (HBV) HBx gene in viral infection. Please note that the title of this grant has been changed from "Transcription and Transformation by Hepatitis B Virus HBx Protein," to" Role of HBx protein in HBV replication" to reflect the progression of our studies on HBx. AIM 1: Molecular mechanism for human hepatitis B virus (HBV) HBx protein action. HBx activates cytoplasmic signal transduction pathways. HBx activation of the Pyk2-Src tyrosine kinase signalling pathway represents an important activity for viral replication in hepatocytic cell lines. Studies are outlined to understand the molecular mechanism for HBx activation of Pyk2-Src signal transduction, since it is tightly linked to HBx stimulation of viral replication. In addition, HBx appears to possess nuclear functions that may stimulate viral transcription. Studies will also investigate the mechanism by which HBx functions in viral transcription, including a possible role in the nucleus, and its impact on viral replication. AIM 2: Role of HBx protein in HBVreplication. Studies are proposed to determine the molecular basis for HBx activation of HBV reverse transcription and DNA replication. Model systems have been developed for delivery of hepadnavirus genomes to differentiated hepatocytic cell lines and rodent primary hepatocytes in culture, that permit viral replication in an HBx-dependent manner. Studies will investigate the role of HBx in viral replication in a biologically relevant system, focusing on HBx induction of HBV core protein phosphorylation, control of the cell cycle, and induction of nucleotide metabolism. HBx-dependent HBV replication will also be studied in primary hepatocytes prepared from mice that are deficient in genes that impact on HBV replication, including knockouts of Src and Pyk2 kinases, to better understand their importance for viral replication.